Detection or detection and imaging of molecules and/or biological processes is an area of scientific and medical importance which is in constant need for innovation. Visual imaging is of particular value to the medical imaging industry and to the pharmaceutical industry. In medical imaging, there is a demand for new imaging agents (contrast agents or diagnostic agents) that enhance the assessment of one or more of healthy tissue, a disease process affecting tissue, and a disease state of affected tissue. As to the pharmaceutical industry, in drug development it is particuarly important to monitor one or more of: (a) the distribuion of the drug in a particular target organ or tissue; (b) the interaction of the drug within the organ or tissue; (c) internalization of the drug by tissue cells, when the target of action is intracellular; and (d) metabolism or bioclearance of the drug in living tissues.
Typically, conventional fluorescent labels (e.g., fluorescein, rhodamine, phycoerythrin, an the like) are used for fluorescence detection and/or imaging. These conventional fluorescent labels typically have an excitation spectrum that may be quite narrow; and hence it is often difficult to find a wavelength spectrum of light suitable for simultaneously exciting several different fluorescent labels (e.g., differing in color of fluorescence emission). However, even when a single light source is used to provide a excitation wavelength spectrum (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different species (e.g., differing in color) of fluorescent labels to permit individual and quantitative detection without substantial spectral overlap. Thus, when using a combination of different fluorescent labels, multiple filters are typically needed to detect the resultant emission spectra of the combination. For example, for fluorescent detection of a substrate labeled with fluoroscein isothiocyanate (FITC), a filter cube comprising a FITC dichromic filter set is used (excitation at 450-490 nm, and peak emission at 520 nm). This example illustrates the current state of the art of fluorescence filter cubes ("fluorescence cubes"). That is, a fluorescence cube is typically designed to ensure that the substrate is excited by a desired short wavelength (specific for the specie of fluorescent label sought to be detected), and detection of wavelengths in a limited spectral band. Table 1 further illustrates the current state of the art of conventional fluorescence cubes comprised of a dichroic mirror (shown is emission wavelength cutoff; i.e., less than the emission wavelength cutoff is reflected away, greater than emission wavelength cutoff is passed onto the barrier filter), an exciter filter (shown is spectrum of incident light), a barrier filter (shown is wavelength cutoff; i.e., less than wavelength cutoff is blocked, greater than wavelength cutoff is passed onto the detector), and fluorochrome detected by the fluorescence cube.
TABLE 1 Dichroic Barrier mirror Exciter filter filter Fluorochrome 400 nm 330 nm-385 nm 420 nm DAPI, Hoechst 33342 455 nm 400 nm-410 nm 455 nm Catecholamine, Serotonin 455 nm 400 nm-440 nm 475 nm Quinacrine 455 nm 420 nm-440 nm 475 nm Acriflavine, Thioflavin S 500 nm 450 nm-480 nm 515 nm FITC 500 nm 470 nm-490 nm 515 nm Acridine orange 570 nm 510 nm-550 nm 590 nm Rhodamine, Propidium iodide, TRITC 600 nm 545 nm-580 nm 610 nm Texas red
Current techniques for acquiring multicolor fluorescence images, using a filter-based imaging method to measure the fluorescence from a substrate labeled with multiple fluorescent labels, are both time-consuming and complicated. Typically, no more than 3 different fluorescent labels may be used due to limitations related to different excitation spectra and different emission spectra. Multicolor fluorescence images are then acquired, one image for each fluorescent label used, by rotating a filter wheel into place. The filter wheel has various filters, wherein each filter or a filter combination is used for a specific fluorescent label (See, e.g., Table 1). Additionally, it is often necessary to make adjustments for each peak wavelength spectrum (i.e., for each label used) such as readjusting the focus of the image, and/or selecting an optimal exposure time for each peak emission spectrum when using a detection system that includes a charge coupling device (CCD) camera. A series of monochrome images are generated, each image corresponding to the peak emission spectrum of a specific fluorescent label. These images are false-colored and then superimposed onto one image using a computer.
Recently, a new class of fluorescent labels have been, and continued to be, developed for use in biological, biomedical, and biochemical applications. These fluorescent labels comprise water-soluble semiconductor nanocrystals ("quantum dots"). "Water-soluble" is used herein to mean sufficiently soluble or suspendable in a aqueous-based solution, such as in water or water-based solutions or physiological solutions, including those used in the various fluorescence detection systems as known by those skilled in the art. Generally, quantum dots can be prepared which result in relative monodispersity; e.g., the diameter of the core varying approximately less than 10% between quantum dots in the preparation. Examples of quantum dots are known in the art to have a core selected from the group consisting of CdSe, CdS, and CdTe (collectively referred to as "CdX"). CdX quantum dots have been passivated with an inorganic coating ("shell") uniformly deposited thereon. Passivating the surface of the core quantum dot can result in an increase in the quantum yield of the fluorescence emission, depending on the nature of the inorganic coating. The shell which is used to passivate the quantum dot is preferably comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se. Typically, CdX core/YZ shell quantum dots are overcoated with trialkylphosphine oxide, with the alkyl groups most commonly used being butyl and octyl. One method to make the CdX core/YZ shell quantum dots water-soluble is to exchange this overcoating layer with a coating which will make the quantum dots water-soluble. For example, a mercaptocarboxylic acid may be used to exchange with the trialkylphosphine oxide coat. Exchange of the coating group is accomplished by treating the water-insoluble quantum dots with a large excess of neat mercaptocarboxylic acid. Alternatively, exchange of the coating group is accomplished by treating the water-insoluble quantum dots with a large excess of mercaptocarboxylic acid in CHCl.sub.3 solution (Chan and Nie, 1998, Science 281:2016-2018). The thiol group of the new coating molecule forms Cd (or Zn)-S bonds, creating a coating which is not easily displaced in solution. Another method to make the CdX core/YZ shell quantum dots water-soluble is by the formation of a coating of silica around the dots (Bruchez, Jr. et al., 1998, Science 281:2013-2015). An extensively polymerized polysilane shell imparts water solubility to nanocrystalline materials, as well as allowing further chemical modifications of the silica surface. Generally, these "water-soluble" quantum dots require further functionalization to make them sufficiently stable in an aqueous solution for practical use in a fluorescence detection system (as described in more detail in U.S. Ser. No. 09/372729, the disclosure of which is herein incorporated by reference), particularly when exposed to air (oxygen) and/or light. Water-soluble functionalized nanocrystals are extremely sensitive in terms of detection, because of their fluorescent properties (e.g., including, but not limited to, high quantum efficiency, resistance to photobleaching, and stability in complex aqueous environments); and comprise a class of semiconductor nanocrystals that may be excited with a single peak wavelength of light resulting in detectable fluorescence emissions of high quantum yield and with discrete fluorescence peaks (e.g., having a narrow spectral band ranging between about 10 nm to about 60 nm).
However, there lacks an optical system comprising a single fluorescence cube which could be used for detecting, or detecting and imaging, (collectively referred to hereinafter as "acquiring") fluorescence spectra generated by a combination of different fluorescent labels used together in multicolor fluorescence analysis of a labeled substrate. More particularly, there is a need for a simple device that enables the simultaneous measurement of the emission spectrum of each of a plurality of different species (e.g., differing in emission spectrum, and hence color) of water-soluble semiconductor nanocrystals which are used in a combination to achieve multicolor fluorescence analysis.